Computational investigation of small RNAs in the establishment of root nodules and arbuscular mycorrhiza in leguminous plants

Many small RNAs have been confirmed to play important roles in the development of root nodules and arbuscular mycorrhiza. In this study, we carried out the identification of certain small RNAs in leguminous plants(Medicago truncatula, soybean, peanut and common bean), such as miRNAs, tRFs and srRNAs, as well as the computational investigation of their regulations. Thirty miRNAs were predicted to be involved in establishing root nodules and mycorrhiza, and 12 of them were novel in common bean and peanut. The generation of tRFs in M. truncatula was not associated with tRNA gene frequencies and codon usage. Six tRFs exhibited different expressions in mycorrhiza and root nodules. Moreover, srRNA^(5.8S) in M. truncatula was generated from the regions with relatively low conservation at the rRNA 3′ terminal. The protein-protein interactions between the proteins encoded by the target genes of miRNAs, tRFs and srRNAs were computed. The regulation of these three types of sRNAs in the symbiosis between leguminous plants and microorganisms is not a single regulation of certain signaling or metabolic pathways but a global regulation for the plants to own growth or specific events in symbiosis.

Anatomical Structure of Root and Root Nodule of Alnus sibirica-Host of Boschniakia rossica

The root of Alnus sibirica is rich in tissue type. Phloem fibers are obvious and Plasmodesmas are well developed. The number of primary xylem bunches are not stable, there are triarch, tetrarch and pentarch types. The perforation plate of vessel is scalariform. The rays types are radial in shape and unicellular in structure. In root nodule, cortex is the best developed structure, the stele is small. The root nodule is branched. In root nodule tissues, actinomyces proliferate luxuriently, the actinomyces body is in different shapes.

African Traditional Vegetables as Agents of Integrated Soil Fertility ManangementmCrotalaria and Amaranth Farming

The knowledge and understanding of African leafy vegetables has diminished over time, but in the recent past year there is more interest in their research for their nutritive and medicinal values. Of value are those that have the potential to ameliorate soil nutrient levels. This is with the background that despite inorganic fertilizers （IF） have a dramatic impact on agriculture in the world, the economic situation in Sub-Saharan African calls for alternative methods to reduce soil fertility degradation. The use of leguminous vegetables may be a solution. The study looks at the potential contribution of a leguminous traditional vegetable-- Crotalaria brevidens to soil nitrogen under various treatments. During the study which was conducted over two seasons, C. brevidens was grown under two treatments of IF and organic fertilizer （OF） with a control in which there was no fertilizer （NF） applied. A non-leguminous vegetable--Amaranthus dubious （Amaranth） was grown under the same treatments for comparison purposes. Soil analysis was done before and after planting in the treatment subplots. Growth parameters were measured every fortnight and these included leaf number, shoot length, dry weight and nodule number for Crotalaria. Results indicated that the leguminous vegetable was not affected by external inputs and there were no significant differences between treatments and control. The non-leguminous vegetables responded well to both IF and OF treatments in all the measured parameters. This underlines the potential for improving soil nitrogen levels using leguminous vegetables, especially as intercrops with the non-leguminous types.

Effect of Inoculation with Arbuscular Mycorrhizal Fungus on Nitrogen and Phosphorus Utilization in Upland Rice-Mungbean Intercropping System

The effect of arbuscular mycorrhiza fungi （AMF） on plant growth and nutrition utilization in upland rice and mungbean intercropping system was studied. A pot experiment was conducted in the greenhouse and AMF colonization rates of rice and mungbean roots, plant nutrient contents, the ability of nitrogen fixation, and nutrient contents changed in the soil were analyzed. The results were obtained as follows： the rates of AMF colonization of rice and mungbean roots were reached to 14.47 and 92.2% in intercopping system, and increased by 4.11 and 11.95% compared with that of in monocropping; the nirtrogen contents of mungbean and rice were increased by 83.72 and 64.83% in shoots, and 53.76 and 41.29% in roots, respectively, while the contents of iron in shoot and root of mungbean were increased by 223.08 and 60.19%, respectively. In the intercropping system with inoculation of AMF, the biomass of mungbean increased by 288.8%. However, the biomass of rice was not significantly changed among all treatments with or without inoculation of AMF recorded. The number and dry weight of nodules were significantly increased either when mungbean was intercropped with rice or inoculated with AMF. When compared with that of monocropping without AMF inoculation, the contents of nitrogen, phosphorus and iron in nodules of intercropping mungbean with inoculation increased by 80.14, 69.54 and 39.62%, respectively. Additionally, intercropping with AMF inoculation significantly increased soil nitrogen content, but reduced soil phosphorus content. We concluded that upland rice-mungbean intercropping system and inoculation with AMF improved the nutrient uptake, the ability of nitrogen fixation and the growth of mungbean.

Determining N supplied sources and N use efficiency for peanut under applications of four forms of N fertilizers labeled by isotope^15N

Rational application of different forms of nitrogen（N） fertilizer for peanut（Arachis hypogaea L.） requires tracking the N supplied sources which are commonly not available in the differences among the three sources：root nodule,soil and fertilizer.In this study,two kinds of peanut plants（nodulated variety（Huayu 22） and non-nodulated variety（NN-1）） were choosed and four kinds of N fertilizers：urea-N（CONH_2-N）,ammonium-N（NH_4~＋-N）,nitrate-N（NO_3^--N） and NH_4~＋ ＋NO_3^--N labeled by^（15）N isotope were applied in the field barrel experiment in Chengyang Experimental Station,Shandong Province,China,to determine the N supplied sources and N use efficiency over peanut growing stages.The results showed that intensities and amounts of N supply from the three sources were all higher at middle growing stages（pegging phase and podding phase）.The accumulated amounts of N supply from root nodule,soil and fertilizer over the growing stages were 8.3,5.3 and 3.8g m^（-2） in CONH_2-N treatment,which are all significantly higher than in the other three treatments.At seedling phase,soil supplied the most N for peanut growth,then root nodule controlled the N supply at pegging phase and podding phase,but soil mainly provided N again at the last stage（pod filling phase）.For the whole growing stages,root nodule supplied the most N（47.8 and 43.0%） in CONH_2-N and NH_4~＋-N treatments,whereas soil supplied the most N（41.7 and 40.9%） in NH_4~＋ ＋NO_3^--N and NO_3^--N treatments.The N use efficiency was higher at pegging phase and podding phase,while accumulated N use efficiency over the growing stages was higher in CONH_2-N treatment（42.2%） than in other three treatments（30.4%in NH_4~＋-N treatment,29.4%in NO_3^--N treatment,29.4%in NH_4~＋ ＋NO_3^--N treatment）.In peanut growing field,application of CONH_2-N is a better way to increase the supply of N from root nodule and improve the N use efficiency.

Enhanced adaptability of Sesbania rostrata to Pb/Zn tailings via stem nodulation

Sesbania rostrata is wellknown for its stem nodulation, but the roles of stem nodulation in root nodulation and adaptation of S. rostrata to Pb/Zn-enriched tailings environment has been poorly understood. We investigated the effects of inoculating （with stem nodule treatment） and non-inoculating （without stem nodule treatment） Azorhizobium caulinodans on the growth, root nodulation, and N fixation of S. rostrata grown on three different types of soil substrata： Pb/Zn tailings, garden soil amended tailings, and garden soil. The results showed that plant height, stem basal diameter, biomass, chlorophyll content, nitrogen content and N-accumulation per plant were 2.3%-4.9%, 2.2%-7.7%, 27.8%-72.2%, 17.1%-23.5%, 12.3%-34.2%, and 43.1%-131.2%, respectively, higher in treatments with stem nodule than those without stem nodule for the same soil substrate. With respect to soil substrata, all measurements had consistently higher values in tailings than in amended tailings and garden soil, indicating that the poorer the soil condition, the greater the contribution of stem nodule. In contrast, the number and fresh weight of root nodules on plants without stem nodule were 6.9-11.6 times and 5.8-29.0 times higher than those with stem nodule, respectively, especially with respect to the plants grew on Pb/Zn tailings. In general, stem nodulation favored plant growth and nitrogen fixation of S. rostrata, but suppressed root nodulation. With the ability of stem and root nodulation, S. rostrata can be used as a pioneer plant species for remediation of Pb/Zn tailings.

Organic matter contribution to soil fertility improvement and maintenance in red Alder (Alnus rubra) silvopastoral systems

An investigation was conducted to quantify fine roots and roots nodules over the four seasons in forestry and agroforestry alder （Alnus rubra） stands in North Wales. Soil samples collected in each season were excavated at three sampling points （0.30 m, 0.57 m and 1.00 m distance from the base of each tree） from nine trees of the agroforestry and forestry plots. Result showed that the density of live fine root had significant differences in between seasons and treatments （P 〈 0.001）. The mean weight density of live fine root over the four seasons in agroforestry and forestry was 0.27±0.01 kg·m^-3 and 0.54±0.03 kg·m^-3, respectively. Weight density of dead root in each system remained constant throughout the year. The mean weight density of dead root was also significantly different （P 〈 0.01） between forestry and agroforestry systems. Weight density of live and dead root nodule was both constant throughout the year and between the different sampling distances. The mean weight densities of live and dead root nodule over the four seasons were 0.09±0.03 kg·m^-3 and 0.05±0.03 kg·m^-3 in agroforestry and 0.08±0.02 kg·m^-3 and 0.03±0.01 kg·m^-3 in the forestry plots, respectively.

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution

Gene innovation plays an essential role in trait evolution.Rhizobial symbioses,the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae,is one of the most attractive evolution events.However,the gene innovations underlying Leguminosae root nodule symbiosis(RNS)remain largely unknown.Here,we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses.We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection.Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways,particular downstream of chalcone synthase(CHS).Among them,Leguminosae-gain typeⅡchalcone isomerase(CHI)could be further divided into CHI1A and CHI1B clades,which resulted from the products of tandem duplication.Furthermore,the duplicated CHI genes exhibited exon–intron structural divergences evolved through exon/intron gain/loss and insertion/deletion.Knocking down CHI1B significantly reduced nodulation in Glycine max(soybean)and Medicago truncatula;whereas,knocking down its duplication gene CHI1A had no effect on nodulation.Therefore,Leguminosae-gain typeⅡCHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence.This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

How many proteins interacting with symbiosis receptor-like kinase （SymRK） involved in nodulation？

Nodule formation is a tightly regulated process that integrates specific signal exchange and coordinated activation of developmental mechanisms to synchronize bacte-rial infection and organ development. Symbiosis receptor kinase （SymRK） is indispensable for symbiotic signal transduction of root nodule symbiosis （RNS） upon stimulation of root cells by microbial signaling molecules. But the protein turnover model of SymRK and the way for nodulation factor signals downstream transduction from SymRK are not clear. Over the past years, a number of proteins interacting with SymRK which required for root nodule symbiosis have been identified. Here we summarized structures and functions of these pro-teins, and concluded that major challenge would be revealing relations between them and the regulation mechanisms of SymRK in nodulation.

Phosphorus and Nitrogen Interactions in Field-Grown Soybean as Related to Genetic Attributes of Root Morphological and Nodular Traits

Two field experiments with different soybean (Glycine max L.) materials were conducted to investigate the interactions between phosphorus (P) and nitrogen (N) as related to the genetic attributes of root morphological and nodular traits. In experiment one, 13 cultivated soybean varieties were grown in a field with relatively low soil P and N availability. P application with 160 kg P/hm2 as triple superphosphate produced a significant simultaneous increase in the content of both P and N in shoot, demonstrating positive P and N interactions. The addition of P also increased root dry weight, root nodule number, nodule mass, nodule size, and nodulation index, but decreased root length and root surface area, indicating that P may affect N nutrition in soybean through a number of root morphological and nodular traits. Interestingly, like P content, N content appeared to be more correlated with root morphological traits (root weight, root length, and root surface area) than with root nodular traits (nodule number, nodule size, nodule mass, and nodulation index) at both P levels, implying that N taken up by the roots may contribute more to the plant N status than biological N2 fixation under the present experimental conditions. In experiment two, 57 soybean lines of a recombinant inbred line (RIL) population derived from a cross between a cultivated variety and a wild genotype were grown on another field site with moderately sufficient P and N levels to further characterize the genetic attributes of root morphological and nodular traits and their relationships with P and N interactions. The results indicated that all morphological and nodular traits measured continually segregated in the RIL population with a normal distribution of the phenotypic values, indicating that these traits are possibly controlled by quantitative trait loci (QTLs). Genetic analysis revealed that all these root traits had relatively low heritabilities (h2b=74.12,70.65,73.76,56.34,52.59, and 52.24 for root weight, root length, root surface area, nodule number, nodule mass, and nodule size, respectively), suggesting that root morphology and nodule formation are influenced greatly by environmental factors. Correlation analysis of the RILs showed that shoot N content was significantly correlated with P content, confirming positive PXN interactions. Similar to experiment one, shoot N content was only significantly correlated with root morphological traits, but not with root nodular traits, again denoting the fact that the N status in soybean could be attributed more to N uptake from the soil than to biological N2 fixation under the present experimental conditions.

Cellular basis of legume-rhizobium symbiosis

The legume-rhizobium symbiosis represents the most important system for terrestrial biological nitrogen fixation on land.Efficient nitrogen fixation during this symbiosis depends on successful rhizobial infection and complete endosymbiosis,which are achieved by complex cellular events including cell-wall remodeling,cytoskeletal reorganizations,and extensive membrane expansion and trafficking.In this review,we explore the dynamic remodeling of the plant-specific cell wall-membrane system-cytoskeleton(WMC)continuum during symbiotic nitrogen fixation.We focus on key processes linked to efficient nitrogen fixation,including rhizobial uptake,infection thread formation and elongation,rhizobial droplet release,cytoplasmic bridge formation,and rhizobial endosymbiosis.Additionally,we discuss the advanced techniques for investigating the cellular basis of root-nodule symbiosis and provide insights into the unsolved mysteries of robust symbiotic nitrogen fixation.

Mechanisms underlying legume-rhizobium symbioses

Legumes,unlike most land plants,can form symbiotic root nodules with nitrogen-fixing bacteria to secure nitrogen for growth.The formation of nitrogen-fixing nodules on legume roots requires the coordination of rhizobial infection at the root epidermis with cell division in the cortex.The nodules house the nitrogen-fixing rhizobia in organelle-like structures known as symbiosomes,which enable nitrogen fixation and facilitate the exchange of metabolites between the host and symbionts.In addition to this beneficial interaction,legumes are continuously exposed to would-be pathogenic microbes;therefore the ability to discriminate pathogens from symbionts is a major determinant of plant survival under natural conditions.Here,we summarize recent advances in the understanding of root nodule symbiosis signaling,transcriptional regulation,and regulation of plant immunity during legume-rhizobium symbiosis.In addition,we propose several important questions to be addressed and provide insights into the potential for engineering the capacity to fix nitrogen in legume and nonlegume plants.

A novel secreted protein, NISP1, is phosphorylated by soybean Nodulation Receptor Kinase to promote nodule symbiosis

Nodulation Receptor Kinase(NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified.Here, we identified a GmNORK-Interacting Small Protein(GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a “DY” consensus motif at its N-terminus.GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP1~(12D) on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP1~(12A).The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation.